ПРИКЛАДНАЯ БИОХИМИЯ И МИКРОБИОЛОГИЯ, 2013, том 49, № 5, с. 499-503
УДК 582.28
NOVEL MUTATIONS IN p-TUBULIN GENE IN Trichoderma harzianum MUTANTS RESISTANT TO METHYL BENZIMIDAZOL-2-YL CARBAMATE
© 2013 M. Li* **, H. Y. Zhang* ***, and B. Liang**
*Fundamental Science on Radioactive Geology and Exploration Technology Laboratory, East China Institute of Technology,
NanChang, Jiangxi, 330013, China **Faculty of Chemistry Biology and Material Sciences, East China Institute of Technology, Fuzhou 344000, China ***College of Life Science, Henan University, Kaifeng 475001, China e-mail: limin_hit@yahoo.com.cn, muzibug@yahoo.com.cn, hypolb@ecit.cn Recieved December 4, 2012
Twelve low resistant (LR) mutants of Trichoderma harzianum with the capability of grow fast at 0.8 p.g/mL methyl benzimidazol-2-yl carbamate (MBC) were obtained using UV mutagenesis. MR and HR mutants which could grow fast at 10 and 100 p.g/mL MBC, respectively, were isolated by step-up selection protocols in which UV-treated mutants were induced and mycelial sector screening was made in plates with growth medium. Subsequently, P-tubulin genes of 14 mutants were cloned to describe the molecular lesion likely to be responsible for MBC resistance. Comparison of the P-tubulin sequences of the mutant and sensitive strains of T. harzianum revealed 2 new MBC-binding sites differed from those in other plant pathogens. A single mutation at amino acid 168, having Phe (TTC) instead of Ser (TCC), was demonstrated for the HR mutant; a double mutation in amino acid 13 resulting in the substitution of Gly (GGC) by Val (GTG) was observed in P-tubulin gene of MR mutant. On the other hand, no substitutions were identified in the P-tubulin gene and its 5'-flanking regions in 12 LR mutants of T. harzianum.
DOI: 10.7868/S0555109913050085
Benomyl and methyl benzimidazol-2-yl carbamate (BBC and MBC), are benzimidazole fungicides widely used in China and worldwide on a large variety of crops to control plant pathogenic fungi [1]. However, many benzimidazole-resistant pathogenic isolates, e.g. Penicillium spp. [2], Colletotrichum gloeosporioides [3], Botrytis cinerea [4], have been detected in field shortly after intensive and exclusive use of these fungicides. In addition, reversion of resistant populations to fungicide sensitive ones has not been observed even after selection pressure has been removed for many years [5]. The appearance of resistance has become an important factor in limiting the efficacy and useful lifetime of fungicides developed at increasingly higher costs. Exploring new chemical fungicides and applying more fungicides in field are needed to control pathogens more efficiently. Stems form a worldwide need to adopt the practice of sustainable agriculture, using strategies that are environment-friendly, less dependent on agricultural chemicals and less damaging to soil and water resources, there has been considerable interest in the utilization of sublethal doses of chemicals with biocontrol strains which are resistant to them, and the information on this kind of integrated control is accumulating more rapidly than on other combinations of control components [6].
To implement integrated control combining bio-control fungi with chemical fungicides, some biocon-trol fungi with fungicide resistant phenotype have been
isolated gradually [7]. Trichoderma genus has been shown effective as biocontrol agent against a range of economically important aerial and soil-borne fungal plant pathogens [8]. Although mutations of Trichoderma spp. resistant to BBC and MBC have already been isolated [9], the obtaining of promising mutants with higher MBC resistant levels than field populations is important for the adoption of these agents with traditional chemical fungicides. Considered from this point, we decided to isolate promising MBC-resistant mutants of T. harzianum for integrated control in field with MBC using UV mutagenesis and step-up selection protocols.
Previous biochemical and genetic analyses have identified microtubules of Saccharomyces cerevisiae as primary benzimidazole binding target [10]. Benzimidazole functioned by inhibit the polymerization of tubulin monomers into functional microtubules, specifically on P-tubulin subunit. In addition, sequence rearrangement in 5'-flanking region of P-tubulin gene has been also observed in Epichloe typhina resistant strain [11]. The aim of the study was to describe molecular lesion likely to be responsible for MBC resistance in T. harzianum. The results would enrich the growing body of knowledge of P-tubulins connected with fungicide resistance and to find possible endogenous selectable markers for T. harzianum.
MATERIALS AND METHODS
Strains and growth conditions. The isolate T. har-zianum was obtained from East China Institute of Technology. The growth potato dextrose agar (PDA) medium contained (g/L): potato infusion — 200, dextrose — 20, agar — 20. The strain was cultured in plates of 9 cm diameter at 25°C for 1 week. Fifteen ml distilled water with 0.005% (v/v) Tween 80 was poured onto the plate to prepare spore suspension (106 cells per ml). Subsequently, 1 mL of spore suspension was inoculated into 100 mL of liquied potato dextrose medium for chromosomal DNA isolation, incubated at 25°C and 200 rpm on an orbital shaker for 48 h and mycelia were harvested.
Induction of T. harzianum MBC-resistant mutant strains. Classical random mutagenesis and selection approach were taken to isolate T. harzianum MBC-re-sistant strains. First, spore suspension (106 CFU/mL) of the wild type was overlaid on PDA plates containing 5, 10, 50, 100 or 500 |g/mL MBC. The plates then were exposed to a 15W UV light sources at a distance of 30 cm for 90 sec. After UV exposure, the plates were incubated in the dark for 5—7 days at 25°C. Viable colonies were isolated and subcultured on a series PDA plates supplied with 0, 1, 5, 10, 50, 100, 500 or 1000 |g/mL MBC. Fungicide concentration that results in 50% mycelia growth inhibition (EC50) and MBC sensitivity were calculated. Genetic stabilities were detected by triple subculturing without the fungicide, followed by retesting for MBC resistance.
Step-up selection protocol was designed to achieve higher level of drug resistance in which UV treatment fungal mutants were induced and subjected to form sectors [12]. Colonized agar plugs, 4 mm in diameter, were transferred from the margin of actively growing cultures of the sensitive and UV-induced resistant isolates, mycelium-side down, onto potato dextrose agar plates supplied with 30, 100, 500 or 1000 |g/mL MBC. The MBC concentrations used for selection were previously found to be highly inhibitory for each tested isolate. Plates were sealed with parafilm, incubated at 25°C in darkness and observed at 6 day intervals for colony diameter and sectoring. EC50 for growth inhibition mycelium and MBC sensitivity of T. harzianum were calculated. Stabilities of the fast growing mycelial sectors were tested as above.
DNA extraction. Mycelium of the sensitive and resistant isolates of T. harzianum were ground to powder in liquid nitrogen. About 100 mg powdered mycelium was added to 600 |L extraction buffer (200 mM Tris-HCl with 25 mM EDTA, 250 mM NaCl and 0.5% SDS, pH 8.0) containing 60 |g proteinase K (Sigma, USA). Following gentle homogenization, the sample was placed at 37°C for 30 min, centrifuged 5 min at 11.000 x g. The upper phase was transferred to a new tube and extracted with phenol-chloroform. The clear supernatant was precipitated with 600 |L isopropanol.
The nucleic acid pellet was washed with 70% (v/v) ethanol, air-dried, resuspended in 500 |L TE buffer (10 mM Tris-HCl with 1 mM EDTA; pH 8.0) containing 10 |g RNAse (Sigma, USA), incubated 30 min at 37°C, extracted with phenol-chloroform and precipitated with isopropanol as above. The final DNA pellet was washed twice with 70% (v/v) ethanol, air-dried, dissolved in nuclease-free water, and stored at — 20°C until use.
Amplification of p-tubulin gene and Hanking regions from MBC-resistant mutants. PCR amplifications were designed to generate P-tubulin gene coding region or its 5'-flanking region, using genomic DNA from variant resistant T. harzianum mutants as templates. Amplification from the sensitive fungal strain used as a control. Primers were designed based on the P-tubulin gene nucleotide sequences reported previously as follows: 5'-ATGCGTGAGATTGTGAGTTCCC-3' (forward) and 5' - TTACTCCTCCTCGTGCTCAGCA-3' (reverse) for the amplification of1.7 kb P-tubulin gene coding region, 5'-AAGCTTCTCATCAGCAAGCTCG-3' (forward) and 5'-GATGGCTAGTGATGATGCTG-GA-3' (reverse) for 1.5 kb 5'-flanking region. Amplified fragments were examined by agarose gel electrophoresis, followed by cloned and sequenced.
RESULTS AND DISCUSSION
Induction of T. harzianum MBC-resistant mutant strains. Four distinct resistance levels were prescribed to classify T. harzianum MBC-resistant isolates according to previous studies [13]: sensitive (S), could grow at 0.1 ^g/mL of MBC, but was completely inhibited at 0.8 ^g/mL; low resistance (LR), could grow fast at 0.8 ^g/mL of MBC but completely inhibited at 10 ^g/mL; moderate resistance (MR), could grow fast at 10 ^g/mL of MBC and slowly at 50 ^g/mL, but completely inhibited at 100 ^g/mL; high resistance (HR), could grow fast at 100 ^g/mL of MBC, partially inhibited at 1000 ^g/mL and even could grow slowly at 1500 ^g/mL.
A series PDA plates amended with 500, 100, 50, 10 or 5 |g/mL MBC were used for the selection of resistant fungal mutants. UV mutagenesis treatment was performed at 500 |g/mL of MBC and no growth was observed at this top concentration after 7 days of incubation at 25°C. In other experiments MBC concentration was 100, 50, 10 or 5 ^g/mL. Twelve colonies were appeared on plates containing 5 |g/mL MBC. The survivors exhibited normal colony morphology and growth rate compared with the sensitive T. harzianum strain when cultured on medium without MBC. The UV-
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